4-vinylcyclohexene

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-03 to 2017-05-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

4-vinylcyclohexene

EC Number:

202-848-9

EC Name:

4-vinylcyclohexene

Cas Number:

100-40-3

Molecular formula:

C8H12

IUPAC Name:

4-ethenylcyclohex-1-ene

Test material form:

liquid

Details on test material:

4-vinylcyclohexene produced by Evonik Degussa GmbH, Batch 117, purity >98.0 % w/w

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

At the start of the exposure and at renewal (0 and 24 hours), samples were taken after preparation of the test item concentrations and analyzed.
At renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.

Test solutions

Vehicle:

no

Details on test solutions:

The stock solution (10.0 mg/L of the test item were weighed out) was freshly prepared with dilution water prior to the start of the exposure (0 hours) and before renewal of the test solutions (24 hours). The stock solution was stirred with a magnetic stirrer at 1100 rpm for 2 hours at room temperature.
Six test item concentrations in a geometric series with a separation factor of 2.2, prepared by dilution of the stock solution 10.0 mg/L with dilution water, were tested as follows:
0.194 - 0.427 - 0.939 - 2.07 - 4.55 - 10.0 mg/L, which corresponds to geometric mean measured concentrations of the test item of:
0.124 – 0.316 – 0.697 – 1.41 – 2.80 – 7.98 mg/L

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system: Daphnia magna STRAUS (Clone 5)
Reason for the selection of the test system: Daphnia magna is the preferred species in accordance with the test guidelines and is bred at the test facility.
Origin: Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
Breeder: Noack Laboratorien GmbH Kathe-Paulus-Str. 1, 31157 Sarstedt, Germany
Culture: In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 oc, in an incubator, 16 hours illumination, light intensity of max. 1500 lx
Culture medium: Elendt M4, according to OECD 202, Annex 3 (2004 ), modified to a total hardness of 160 to 180 mg CaC03/l, is used.
Culture feeding: The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriela subcapitata and Desmodesmus subspicatus, with an algae cell density of> 10E6 cells/ml. The algae are cultured at the test facility.
Origin of the food algae: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches lnstitut der Universitat Gottingen, Nikolausberger Weg 18, 37073 Gottingen, Germany

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

Test temperature (target) 18- 22 °C, constant within ± 1 °C
During the test period, the temperature in the incubator was 19 – 20 °C.

pH:

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits.

Water Quality Parameters in fresh Media at the Start of the Exposure and at Renewal (0 and 24 hours)

Geometric mean 0 hours 24 hours
measured pH-value Dissolved pH-value Dissolved
concentration O2 concentration O2 concentration
of test item [mg/L] [mg/L]
[mg/L]
7.98 7.61 8.98 7.72 8.89
2.80 7.61 8.96 7.72 8.76
1.41 7.54 8.94 7.69 8.84
0.697 7.51 8.95 7.69 8.83
0.316 7.41 8.93 7.65 8.79
0.124 7.44 8.95 7.63 8.66
Control 8.31 8.89 7.59 8.92

Dissolved oxygen:

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits.

Water Quality Parameters in fresh Media at the Start of the Exposure and at Renewal (0 and 24 hours)

Geometric mean 0 hours 24 hours
measured pH-value Dissolved pH-value Dissolved
concentration O2 concentration O2 concentration
of test item [mg/L] [mg/L]
[mg/L]
7.98 7.61 8.98 7.72 8.89
2.80 7.61 8.96 7.72 8.76
1.41 7.54 8.94 7.69 8.84
0.697 7.51 8.95 7.69 8.83
0.316 7.41 8.93 7.65 8.79
0.124 7.44 8.95 7.63 8.66
Control 8.31 8.89 7.59 8.92

Nominal and measured concentrations:

The measured concentrations of the test item were in the range of 64 to 97% of the nominal values in fresh media (0 and 24 hours) and 51 to 80% in 24-hour old media (24 and 48 hours). The geometric mean measured concentrations of the test item were: 0.124 – 0.316 – 0.697 – 1.41 – 2.80 – 7.98 mg/L.
see table below " overall remarks"

Details on test conditions:

Stock solution: The stock solution (10.0 mg/L of the test item were weighed out) was freshly prepared with dilution water prior to the start of the exposure (0 hours) and before renewal of the test solutions (24 hours). The stock solution was stirred with a magnetic stirrer at 1100 rpm for 2 hours at room temperature.

Test concentrations: Six test item concentrations in a geometric series with a separation factor of 2.2, prepared by dilution of the stock solution 10.0 mg/L with dilution water, were tested as follows:
0.194 - 0.427 - 0.939 - 2.07 - 4.55 - 10.0 mg/L, which corresponds to geometric mean measured concentrations of the test item of:
0.124 – 0.316 – 0.697 – 1.41 – 2.80 – 7.98 mg/L
The test item concentrations mentioned above were selected based on the results of a non-GLP preliminary range finding test. For results, see below

Control: Dilution water without test item incubated under the same conditions as the test groups.

Test method: The study was performed with a semi-static test design. Due to the volatility of the test item, the study was performed in a closed system without headspace according to OECD guidance document no. 23, ENV/JM/MONO(2000)6 to reduce contact with air and losses of the test item by evaporation.

Test duration: 48 hours

Test vessels / volume: Sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps (made from polypropylene) were used and filled up to the top with the test solutions. A test volume of approximately 130 mL was provided in each test vessel.

Dilution water: Same composition as the culture medium

Number of daphnids and replicates: 20 daphnids, divided into 4 replicates, each with 5 daphnids, were and replicates used per concentration level and control.

Age of the daphnids at the start of the exposure: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.
 
Acclimatization: Acclimatization of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.

Application: The test vessels were filled up to the top with the test solutions. There was no headspace in the test vessels. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (renewal of the test solutions) by pipette. Thereafter, the test vessels were closed immediately with screw caps.

Renewal of the test solutions: The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled up to the top with the freshly prepared test solutions and the daphnids were transferred by pipette.

Illumination (target) Diffuse light, light intensity of max. 1500 lx

Photoperiod (target) 16/8 hours light/dark cycle

Feeding The daphnids were not fed during the study.


Results of the non-GLP Preliminary Range Finding Test:
A non-GLP preliminary range finding test under semi-static conditions over a period of 48 hours was conducted at the test facility in a closed system without headspace with three nominal test item concentrations of 0.5, 5 and 50 mg/L. The test item concentrations were prepared as specified for the definitive test. All concentration levels were visually clear throughout the exposure period. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). In the range finding test, two replicates per treatment group and control, each with ten daphnids, were tested. The concentrations of the test item were analytically verified via GC-MS.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

other: EC 10 / 50 /100

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: > 100 mg/L main component

Effect concentrations 2

Duration:

24 h

Dose descriptor:

other: EC 10 / 50 /100

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: > 100 mg/L main component

Details on results:

Based on the geometric mean measured concentrations of the test item, the 48 hours EC50 for Daphnia magna was 2.88 mg/L (95% confidence limits: 1.52 – 5.46 mg/L).

EC10-, EC50- (with 95% Confidence Limits) and EC100-Values (based on the geometric mean measured concentrations of the test item)

Effect concentrations Test duration [hours] Geometric mean measured test item concentration [mg/L]
EC10
(with 95% confidence limits) 24 2.66 (Cl: 1.57 – 2.80)
48 2.68 (Cl: 1.45 – 5.35)
EC50
(with 95% confidence limits) 24 3.38 (Cl: 2.83 – 6.27)
48 2.88 (Cl: 1.52 – 5.46)

EC100 24 7.98
48 7.98




see table below "any other information on results"

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2017 04-06 to 2017-04-07.

Current Study Valid Range
EC50 1.72 mg/L 0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
95% confidence limits 1.34 - 1.93 mg/L 0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A

Any other information on results incl. tables

 Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test

           (n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured concentration of the test item [mg/L]	IMMOBILIZATION [%]
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	Mean	1	2	3	4	Mean
7.98	100	100	100	100	100	100% mortality after 24 hours
2.80	0	20	20	20	15	20	40	40	20	30
1.41	0	0	0	0	0	0	0	0	0	0
0.697	0	0	0	0	0	0	0	0	0	0
0.316	0	0	0	0	0	0	0	0	0	0
0.124	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Absolute Numbers of immobileDaphnids after 24 and 48 hours ofExposure in the Definitive Test

           (n = 20, divided into 4 replicates with 5 daphnids each)

Geometric mean measured concentration of the test item [mg/L]	Number of immobile Daphnids / Total number of Daphnids
	24 h					48 h
	Replicates					Replicates
	1	2	3	4	Mean	1	2	3	4	Mean
7.98	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20	5 / 5	5 / 5	5 / 5	5 / 5	20 / 20
2.80	0 / 5	1 / 5	1 / 5	1 / 5	3 / 20	1 / 5	2 / 5	2 / 5	1 / 5	6 / 20
1.41	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
0.697	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
0.316	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
0.124	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20
Control	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20	0 / 5	0 / 5	0 / 5	0 / 5	0 / 20

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item, the 48 hours EC50 for Daphnia magna was 2.88 mg/L (95% confidence limits: 1.52 – 5.46 mg/L).

Executive summary:

In the acute immobilization test with Daphnia magna (STRAUS), the effects of the test item were determined from 2017‑05-03 to 2017-05-05 at the test facility according to OECD 202 (2004), which is equivalent to the Commission Regulation (EC) No.440/2008 Method C.2 (2008).

The study was conducted in a closed system (sealed glass flasks) without headspace, due to the volatility of the test item, to reduce contact with air and losses of the test item by evaporation. The study was performed under semi-static conditions over a period of 48 hours with six test item concentrations in the range of 0.194 to 10.0 mg/L in a geometric series with a separation factor of 2.2.

The test item is a colorless and clear liquid with a water solubility of 50 mg/L (at 25 °C). A stock solution of 10.0 mg/L of the test item was prepared in dilution water as specified. The stock solution was stirred with a magnetic stirrer at 1100 rpm for 2 hours at room temperature. The stock solution was used as the highest concentration level and for the preparation of further test concentrations by dilution with dilution water.

All concentration levels0.194 to 10.0 mg/Lwere visually clear throughout the exposure period.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each test item concentration and the control.

The concentrations of the test item were analytically verified via GC-MS in fresh media at the start of the exposure and at renewal (0 and 24 hours) as well as in 24-hours old media at renewal and at the end of the test (24 and 48 hours) at all concentration levels and in the control.

The measured concentrations of the test item were in the range of 64 to 97% of the nominal values in fresh media (0 and 24 hours) and 51 to 80% in 24-hour old media (24 and 48 hours). The geometric mean measured concentrations of the test item were: 0.124 – 0.316 – 0.697 – 1.41 – 2.80 – 7.98 mg/L.

Since the measured test item concentrations were not within ± 20% of the nominal concentrations, all effect concentrations given in are based on the geometric mean measured concentrations of the test item.

The validity criteria of the test guideline were fulfilled.

 

Conclusion

Based on the geometric mean measured concentrations of the test item, the 48 hours EC50 for Daphnia magna was 2.88 mg/L (95% confidence limits: 1.52 – 5.46 mg/L).